Synthesis and Physicochemical Characterization of Folate−Cyclodextrin Bioconjugate for Active Drug Delivery

β-Cyclodextrin−poly(ethylene glycol)−folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure:  (1) synthesis of CD-PEG-NH<INF>2</INF> by reaction of monotosyl-activated β-cyclodextrin with excess of 700 Da diamino-PEG; (2) synthesis of CD-PEG-FA by reaction of CD-PEG-NH<INF>2</INF> with succinimidyl ester-activated folic acid. The CD-PEG-NH<INF>2</INF> intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH<INF>2</INF> and CD-PEG-FA were analyzed by mass spectrometry, ¹H NMR, and UV−vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates:  the CD-PEG-NH<INF>2</INF> intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of β-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified β-cyclodextrin (16.3 mM). Phase solubility diagrams of β-estradiol revealed that drug solubility increases from 11 μM in buffer to 600 μM in the presence of β-cyclodextrins and 5900 μM with CD-PEG-FA. However, the affinity of β-estradiol for β-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with β-cyclodextrin. Computer modeling studies showed that the folic acid linked to the β-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified β-cyclodextrin.